Methods of making high-strength NDGA polymerized collagen fibers and related collagen-prep methods, medical devices and constructs

ABSTRACT

The disclosure describes methods of making high-strength NDGA collagen and associated methods of preparing collagen preparatory material and medical bioprostheses.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/964,756, filed Dec. 27, 2007, which claims the benefit of priority toU.S. Provisional Application Ser. No. 60/882,065, filed Dec. 27, 2006,U.S. Provisional Application Ser. No. 60/883,408, Filed Jan. 4, 2007,and U.S. Provisional Application No. 60/890,660, filed Feb. 20, 2007,the contents of which are hereby incorporated by reference as if recitedin full herein.

FIELD OF THE INVENTION

The invention relates to biomedical materials.

BACKGROUND OF THE INVENTION

Koob et al. have described methods of producing nordihydroguaiareticacid (NDGA) polymerized collagen fibers of tensile strengths similar tothat of natural tendon (e.g., about 91 MPa) to make medical constructsand implants. See, Koob and Hernandez, Material properties ofpolymerized NDGA-collagen composite fibers: development of biologicallybased tendon constructs, Biomaterials 2002 January; 23 (1): 203-12, andU.S. Pat. No. 6,565,960, the contents of which are hereby incorporatedby reference as if recited in full herein.

SUMMARY OF EMBODIMENTS OF THE INVENTION

Embodiments of the present invention are directed to improved methods ofproducing biocompatible NDGA polymerized fibers. Some embodiments aredirected at producing high-strength NDGA polymerized fibers used to makeimplantable biocompatible constructs, implants and/or other prostheses.

Some embodiments are directed to methods of manufacturingnordihydroguaiaretic acid (NDGA) polymerized collagen fibers. Themethods include: (a) treating collagen with a solution comprising NDGA;(b) drying the NDGA treated collagen while holding the collagen intension for a period of time; (c) washing the dried NDGA treatedcollagen in a solution to remove unreacted soluble NDGA cross-linkingintermediates; (d) drying the NDGA treated collagen while holding thecollagen in tension for a period of time; and (e) repeating steps(a)-(d) at least once to produce high-strength NDGA polymerized collagenfibers.

In some embodiments, the methods can also include, after steps (a)-(d)are repeated at least once, forming a bioprosthesis using the highstrength NDGA polymerized fibers. In some embodiments, the bioprosthesiscan be a ligament bioprosthesis that has a tensile strength of betweenabout 180-280 MPa, and a stiffness and dynamic flexibility that meets orexceeds that of a natural ligament. In other embodiments, thebioprosthesis can be a tensile strength between about 180-280 MPa, and astiffness and dynamic flexibility that meets or exceeds that of anatural tendon.

Still other embodiments are directed to biomedical implants. Theimplants include at least one high-strength synthetic NDGA polymerizedcollagen fiber.

In some embodiments, the at least one fiber is a plurality of fibers,and the bioprosthesis is a ligament bioprosthesis that has a tensilestrength of between about 180-300 MPa, and a stiffness and dynamicflexibility that meets or exceeds that of a natural ligament. In otherembodiments, the at least one fiber is a plurality of fibers, and thebioprosthesis has a tensile strength between about 180-300 MPa, and astiffness and dynamic flexibility that meets or exceeds that of anatural tendon.

Yet other embodiments are directed to medical kits for a tendon orligament repair, augmentation or replacement. The kits include ahigh-strength NDGA collagen fiber construct and a sterile packagesealably enclosing the NDGA collagen fiber construct therein.

Among other things, the NDGA collagen fiber construct can be a ligamentbioprosthesis that has a tensile strength of between about 180-300 MPa atendon bioprosthesis that has a tensile strength of between about180-300 MPa.

Still other embodiments are directed to medical kits that include animplantable medical device comprising NDGA collagen fiber derived fromechinoderm collagen; and a sterile package sealably enclosing the devicetherein. The NDGA collagen fibers may have an average tensile strengthof about 100 MPa.

Additional embodiments are directed to medical kits that include adevice comprising NDGA collagen fiber derived from porcine collagen anda sterile package sealably enclosing the device therein. The NDGAcollagen fibers may be high-strength fibers.

Other embodiments are directed to medical kits that include a devicecomprising NDGA collagen fiber derived from caprine collagen and asterile package sealably enclosing the device therein. The NDGA collagenfibers may be high-strength fibers.

Other embodiments are directed to methods of organizing collagen beforecross-linking. The methods include: (a) purifying donor collagenpreparatory material; (b) dialyzing the purified collagen preparatorymaterial a plurality of times; and (c) forming a substantially clear gelusing the dialyzed collagen material thereby indicating improvedorganization of collagen fibrils.

The dialyzing can be carried out three times against dionized water (DI)in a volume ration of between about 30:1 to about 100:1, for betweenabout 30-90 minutes. Typically, each dialyzing is carried out againstdionized water (DI) in a volume ration of about 60 to 1 for about 40minutes.

Further features, advantages and details of the present invention willbe appreciated by those of ordinary skill in the art from a reading ofthe figures and the detailed description of the embodiments that follow,such description being merely illustrative of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart of operations that can be used to carry outembodiments of the invention.

FIG. 2 is a flow chart of operations that can be used to carry outembodiments of the invention.

FIG. 3 is a flow chart of operations that can be carried out beforecross-linking for improved organization of collagen fibrils in collagenpreparatory material according to embodiments of the invention.

FIG. 4 is a schematic illustration of an NDGA-treated fiber held intension during a drying operation according to embodiments of thepresent invention.

FIG. 5 is a schematic illustration of a medical kit comprising ahigh-strength NDGA-treated collagen construct according to embodimentsof the invention.

FIG. 6 is a graph of tensile strength (MPa) of high strength NDGA fibersrelative to other fibers, including prior NDGA fibers, according toembodiments of the invention.

FIG. 7 is a graph of tensile strength (MPa) of fibers using collagenfrom different sources according to embodiments of the invention.

DETAILED DESCRIPTION

The present invention now is described more fully hereinafter withreference to the accompanying drawings, in which embodiments of theinvention are shown. This invention may, however, be embodied in manydifferent forms and should not be construed as limited to theembodiments set forth herein; rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Like numbers refer to like elements throughout. In the figures, thethickness of certain lines, layers, components, elements or features maybe exaggerated for clarity. Broken lines illustrate optional features oroperations unless specified otherwise.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items. As used herein, phrases such as “between X and Y” and“between about X and Y” should be interpreted to include X and Y. Asused herein, phrases such as “between about X and Y” mean “between aboutX and about Y.” As used herein, phrases such as “from about X to Y” mean“from about X to about Y.”

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the specification andrelevant art and should not be interpreted in an idealized or overlyformal sense unless expressly so defined herein. Well-known functions orconstructions may not be described in detail for brevity and/or clarity.

It will be understood that when an element is referred to as being “on”,“attached” to, “connected” to, “coupled” with, “contacting”, etc.,another element, it can be directly on, attached to, connected to,coupled with or contacting the other element or intervening elements mayalso be present. In contrast, when an element is referred to as being,for example, “directly on”, “directly attached” to, “directly connected”to, “directly coupled” with or “directly contacting” another element,there are no intervening elements present. It will also be appreciatedby those of skill in the art that references to a structure or featurethat is disposed “adjacent” another feature may have portions thatoverlap or underlie the adjacent feature.

It will be understood that, although the terms first, second, etc. maybe used herein to describe various elements, components, regions, layersand/or sections, these elements, components, regions, layers and/orsections should not be limited by these terms. These terms are only usedto distinguish one element, component, region, layer or section fromanother region, layer or section. Thus, a first element, component,region, layer or section discussed below could be termed a secondelement, component, region, layer or section without departing from theteachings of the present invention. The sequence of operations (orsteps) is not limited to the order presented in the claims or figuresunless specifically indicated otherwise.

The terms “implant” and “prosthesis” are used interchangeably herein todesignate a product configured to repair or replace (at least a portionof) a natural tendon, ligament or other tissue of a mammalian subject(for veterinary or medical (human) applications). The term “implantable”means the device can be inserted, embedded, grafted or otherwisechronically attached or placed on or in a patient. The term “agitate”and derivatives thereof refer to mixing the components in a vessel bymoving, shaking, vibrating, oscillating, rotating, centrifuging or othermovement types, including combinations of the movement types.

The term “dynamic flexibility” means that the bioprosthesis is able toperform at least as well as the target tissue undergoing repair, such asa natural ligament or tendon, so as to be able to dynamically stretchand compress, and typically allow some torsion, to behave at least aswell as the repaired or replaced target tissue.

The collagen can be of any form and from any origin. The collagen can beany of the identified collagen genotypes, for example, the interstitialfiber forming collagen types I, II and III, as well as any othersubstantially fiber forming types of collagen, for example collagen VI.The collagen can be acid soluble collagen or pepsin solubilizedcollagen. The collagen can be from mammalian cells synthesized in vitro.The collagen can be from molecularly engineered constructs andsynthesized by bacterial, yeast or any other molecularly manipulatedcell type. For example, the collagen can be sea cucumber dermiscollagen, bovine, caprine, porcine, ovine or other suitable donormammal, marine animal collagen such as chinoderms, molecularlyengineered collagen, or gelatin (e.g., in any suitable form includingsolid, gel, hydrogels, liquids, or foams). In addition, the collagen canbe digested with a protease before the oxidizing and polymerizing steps.The collagen can be in the form of microfibrils, fibrils, naturalfibers, or synthetic fibers. The polymeric material, e.g., collagen, canbe solubilized, dissolved or otherwise transferred into an acidsolution, for example, acetic acid (e.g., about 0.01M to about 1.0M,typically about 0.5M), hydrochloric acid (between about pH 1 to about pH3, typically about pH 2.0), or any other suitable acid at appropriateconcentration (e.g., about pH 1.0 to about pH 3.0, typically about pH2.0). The collagen can also be dissolved in a neutral buffered solutioneither with or without salts, e.g., phosphate buffer at about pH 7.0,phosphate buffered saline at about pH 7.0. The phosphate buffer can beat any concentration of sodium phosphate between about 0.01 and 0.5, butmore typically between about 0.02 and about 0.1M. The buffer can also beany buffer, including, but not limited to, sodium acetate, HEPES, orMOPS. The collagen can be present in a quantity that is at least about0.1% to about 10%, typically between 0.1% to about 5% (e.g, about 0.1,0.2, 0.3, 0.4, 1.0, 2.0, 4.0%) by weight per volume before dialyzing, orby weight per volume in the neutral buffer solution beforefibrillogenesis and fiber formation. In the dried fiber, collagen can bebetween about 50-100% (e.g., at least about 75%, 90%, 95% or 100%)before crosslinking.

Collagen “microfibrils,” “fibrils,” “fibers,” and “natural fibers” referto naturally-occurring structures found in a tendon. Microfibrils areabout 3.5 to 50 nm in diameter. Fibrils are about 50 nm to 50 μm indiameter. Natural fibers are above 50 μm in diameter. A “syntheticfiber” refers to any fiber-like material that has been formed and/orchemically or physically created or altered from its naturally-occurringstate. For example, an extruded fiber of fibrils formed from a digestedtendon is a synthetic fiber but a tendon fiber newly harvested from amammal is a natural fiber. Of course, synthetic collagen fibers caninclude non-collagenous components, such as particulates, hydroxyapatiteand other mineral phases, or drugs that facilitate tissue growth orother desired effects. See, U.S. Pat. No. 6,821,530, incorporated hereinby reference above. For example, the fibers and/or constructs formedfrom same, can include compositions that can contain carbon nano-tubes,zinc nano-wires, nano-crystalline diamond, or other nano-scaleparticulates; and larger crystalline and non-crystalline particulatessuch as calcium phosphate, calcium sulfate, apatite minerals. Forexample, the compositions can contain therapeutic agents such asbisphosphonates, anti-inflammatory steroids, growth factors such asbasic fibroblast growth factor, tumor growth factor beta, bonemorphogenic proteins, platelet-derived growth factor, and insulin-likegrowth factors; chemotactic factors such fibronectin and hyaluronan; andextracellular matrix molecules such as aggrecan, biglycan, decorin,fibromodulin, COMP, elastin, and fibrillin. In some embodiments, thefibers and/or fiber-derived constructs can contain cells, engineeredcells, stem cells, and the like. Combinations of the above or othermaterials can be embedded, coated and/or otherwise attached to thefibers and/or construct formed from same.

Properly processed NDGA polymerized fibers are biocompatible asdiscussed in U.S. Pat. No. 6,565,960, incorporated by referencehereinabove. FIG. 1 illustrates operations that can be used to formhigh-strength collagen fibers. The term “high-strength” refers to fibershaving an average tensile strength of at least about 150 MPa, such asbetween about 180 MPa and 350 MPa, and typically, for bovine, porcine orcaprine based “donor” collagen, between about 180 MPa and 280 MPa, suchas about 279 MPa (measured on average). The fibers may also havesuitable stiffness and strain yield. In general, the fibers formed fromthe compositions and processes of the invention can have a stiffness ofat least about 200 MPa (e.g., at least about 300, 400, 500, or 600 MPa),and a strain at failure of less than about 20% (e.g., less than about 15or 10%). The fibers may be formed with a relatively thin diameter, suchas, for example about a 0.08 mm dry diameter (on average) and about a0.13 mm wet diameter (on average).

To measure these physical properties, any suitable apparatus having (1)two clamps for attaching to the fiber(s), (2) a force transducerattached to one of the clamps for measuring the force applied to thefiber, (3) a means for applying the force, and (4) a means for measuringthe distance between the clamps, is suitable. For example, tensiometerscan be purchased from manufacturers MTS, Instron, and Cole Farmer. Tocalculate the tensile strength, the force at failure is divided by thecross-sectional area of the fiber through which the force is applied,resulting in a value that can be expressed in force (e.g., Newtons) perarea. The stiffness is the slope of the linear portion of thestress/strain curve. Strain is the real-time change in length during thetest divided by the initial length of the specimen before the testbegins. The strain at failure is the final length of the specimen whenit fails minus the initial specimen length, divided by the initiallength.

An additional physical property that is associated with the extent ofcross-linking in a composition is the shrinkage temperature. In general,the higher the temperature at which a collagenous composition begins toshrink, the higher the level of cross-linking. The shrinkage temperatureof a fiber can be determined by immersing the fiber in a water or bufferbath, raising the temperature of the water or buffer bath, and observingthe temperature of the water or buffer bath at which the fiber shrinks.Tension on the fiber may be required for observing the shrinkage. Theshrinking temperature for the compositions of the invention can be atleast about 60 degrees C. (e.g., at least 65 or 70 degrees C.).

For compositions that are not elongated in shape, such as in a disk, thefracture pressure in compression loading can be an indication ofphysical strength. The fracture pressure is the minimum force per areaat which a material cracks.

It is believed that high-strength fibers allow for improved oralternative bioprosthesis constructs and/or medical devices. Forexample, high-strength fibers may be particularly suitable forbioprostheses suitable for tendon and/or ligament repair, augmentation,and/or replacement. A biomaterial with increased strength over that ofnatural tissue (muscle and the like) can allow for a bioprosthesis thathas a smaller cross-sectional area than that of the natural tissue beingreplaced or repaired. The smaller area can improve the function of thebioprosthesis as a scaffold for neo-tendon or ligament in-growth, whichmay augment strength and/or long term survival rate of the repair. Theuse of high-strength fibers on medical devices and constructs may alsooffset or reduce the effects of stress concentration factors that resideat regions of integration in adjacent tissue such as bone.

Referring to FIG. 1, some methods include obtaining or harvestingpepsin-solubilized collagen from a donor source. The harvested collagencan be treated using a solution comprising NDGA to polymerize thecollagen (block 10). The NDGA treated collagen can then be dried whilethe collagen is held in tension for a desired period of time (block 20).The typical tension force during at least part of the drying operationis between about 2-4 grams weight per fiber. The “dried” collagen canthen be placed in a liquid bath or solution (typically an ethanolsolution) and washed to remove any unreacted soluble NDGA cross-linkingintermediates (block 30). That is, after the NDGA polymerizationprocess, the NDGA treated collagen fibers can be washed in an ethanolsolution (typically including phosphate buffered saline) to removepotential cytotoxins due to leachable reaction products. After washing,the collagen can then be dried again while held in tension (block 40).This sequence can be repeated at least once (block 50); typically onlytwo repetitions are needed to achieve the desired tensile strength.

The drying may be at room temperature, typically at between about 50° F.(10° C.) to about 80° F. (27° C.) or may be carried out at suitable, lowheating temperatures, below about 105° F. (40.6° C.), with or with outthe aid of forced gas flow (e.g., fans to blow air). Different dryingtimes and temperatures may be used during a single drying event orbetween drying events. The drying can be carried out in a sterile and/orsuitable clean-room environment and/or sterilized after the process iscompleted before or after packaging. The collagen may be partially orsubstantially totally dried. In some embodiments, the collagen is notrequired to be completely dry before the next step. The desired periodof drying time can be between about 1-5 hours, typically about 2 hoursfor a typical amount of collagen (block 22). The washing can includeagitating the NDGA-treated collagen in a solution of between about50-95% ethanol, typically about 70% ethanol, in an amount of at leastabout 50 ml of 70% ethanol per gram of dry fiber.

The tensile force can be provided as shown in FIG. 4, by clamping,pinching, rolling or otherwise attaching one end portion 200 e ₁ of afiber 200 to a rod or other holding member 205 and attaching at leastone weight 210 to an opposing end portion 200 e ₂. A single weight maybe used for more than one fiber or each fiber may use more than oneweight. Other tensioning mechanisms or configurations may also be used.Substantially horizontal, angled or other non-vertically orientedtensioning systems may be used. In some embodiments, weights can beapplied to both opposing end portions of the fiber(s) to generate thedesired tension. A typical weight of about 2-10 grams per fiber,depending on the extruded fiber size, can be appropriate.

FIG. 2 illustrates operations that can be used to form NDGA-treatedcollagen fibers according to other embodiments of the invention. Asshown, donor collagen material is obtained and purified as appropriate(block 100). The donor material can be from any suitable source. FIG. 6illustrates different fiber tensile strengths (average) obtained usingdifferent donor collagen sources. The purified collagen preparatorymaterial is dialyzed, incubated, then placed in a fiber-forming bufferthat is then extruded (block 105).

FIG. 3 illustrates operations that can be used to form improvedorganization of collagen fibrils using a dialyzing process. As notedabove, preparatory donor collagen material can be purified (block 60).The purified collagen preparatory material is dialyzed a plurality oftimes in a selected liquid for a desired period of time (block 65). Thedialyzing is typically repeated three times (block 72). The dialyzingcan be carried out against dionized (DI) water in a volume ratio ofbetween about 30:1 to about 100:1, typically about 60 to 1, for betweenabout 30-90 minutes, typically about 40 minutes (block 66). Thedialyzing can form a substantially clear gel of collagen fibrilsindicating good organization (block 70), where opacity indicates lessorganization. The organization can help improve tensile strength ofsubsequently cross-linked fibers.

The dialyzed collagen material can be incubated for a desired timebefore placing in a fiber-forming buffer (block 75). The dialyzed gelcan be cross-linked to provide collagen fibers for medical constructs(block 76). The polymerization (e.g., cross-linking) can be carried outusing NDGA and the resultant NDGA treated collagen fibers can berelatively thin, such as, for example, about 0.08 mm dry diameter (onaverage) (block 77).

The incubation may be for at least about 24 hours, typically 24-48hours, and may be at room temperature of between about 15-30° C.,typically about 25° C. The dialysis process can be used beforecross-linking for subsequent use with any suitable cross-linkingmaterials, to promote collagen organization, such as, for example, andthe process is not limited to NDGA, but may be useful with othermaterials, including, for example, glutaraldehyde. The dried collagenfiber can also be treated with other methods to improve the tensileproperties of the fiber. The dried collagen fibers produced by themethod(s) described herein can be cross-linked with agents such asglutaraldehyde, formaldehyde, epoxy resins, tannic acid, or any otherchemical agent that produces covalent cross-links between collagenmolecules within fibrils or between fibrils. Alternatively, the driedfiber can be treated to induce cross-linking between collagen moleculessuch as, but not limited to, one or more of a carbodiimide treatment,ultraviolet irradiation either with or without carbohydrates to initiateglycation adducts, and dehydrothermal treatment coupled with any of theaforementioned methods.

The fiber-forming buffer can include about 30 mM NaH₂PO₄, 140 mM NaCl,in a volume ratio of about 60 to 1, for between about 12-24 hours,typically about 16 hours at a slightly elevated temperature of about 37°C. The extrusion can be directed to enter directly or indirectly into anaqueous bath, such as a water or saline bath, and hung from one endportion. To remove from the bath, the extruded material can be liftedout of the bath at a slow rate of less than about 5 mm/min, typicallyabout 1 mm/min. The extruded fibers can then be dried (block 110). Todry, the fibers may be hung or otherwise held for at least about 5hours, typically for at least about 6 hours, such as between about 6-10hours.

Referring again to FIG. 2, as shown, the extruded dried fibers can behydrated in a liquid buffer solution (block 120). The hydration can befor between about 30 minutes to about 3 hours, typically about 1 hour,in a solution of at least 50 ml of buffer (such as 0.1 M NaH₂PO₄, pH7.0) per gram of dry fiber. In some embodiments, the pH of the phosphatebuffered solution can be increased to above pH 7 to a pH of about 11 orbetween 7-11, e.g., a pH of about 8.0, 9.0, 10.0 or 11.0.

The hydrated fibers in the buffer solution can then be combined with aliquid solution comprising (dissolved) NDGA (block 130). About 30 mg/mlof the NDGA can be dissolved in about 0.4 NaOH prior to combining withthe buffer/fiber solution. The dissolved NDGA solution can be added inan amount of between about 3-4 mg NDGA per ml of buffer, such as about0.1 M NaH₂PO₄. The NDGA and fiber solution can be agitated, shaken,centrifuged, rotated, oscillated or otherwise moved and/or mixed for alength of time (block 140), typically between about 12-48 hours, such asat least about or about 16 hours. As discussed above with respect toFIG. 1, the fibers can then be removed and held in tension (e.g., hungor stretched), during a drying operation (block 150), typically lastingat least about 2 hours. The (partially or wholly) dried fibers can thenbe washed to remove unwanted reaction products (block 160). Typically,the fibers are washed (agitated) in about 70% ethanol as also discussedabove, then held in tension during a drying operation (block 170). Thesteps 120-170 can be repeated (block 175).

FIG. 5 illustrates a medical kit 250 that includes a medical device orimplant 225 with at least one NDGA-treated collagen fiber 200. The kit250 may include other components, such as, for example, a container ofsurgical adhesive, sutures, suture anchors, and the like. The device orimplant 225 may be held hydrated in a flexible sealed package of sterileliquid 230. The kit 250 may include a temperature warning so that theconstruct 225 is not exposed to unduly hot temperatures that may degradethe implant. A temperature sensor may optionally be included on thepackage of the kit (not shown) to alert the clinician as to anyexcessive or undue temperature exposure prior to implantation. Forexample, it may be desirable to hold or store the kit 250 (and implantor device 225) at a temperature that is less than about 37° C. and/or100° F. prior to implantation. The kit 250 may be packaged in a housingwith a temperature controlled or insulated chamber 250 c to facilitatean appropriate temperature range.

Embodiments of the invention can form implants and/or medical devicesusing NDGA collagen fibers with different tensile strengths from asingle source type, e.g., NDGA-treated bovine collagen, with both lowstrength, such as less than about 90 MPa tensile strength, typicallybetween about 10 MPa and 90 MPa, and high strength fibers and/or usingNDGA-treated collagen from more than one source type (e.g., bovine andechinoderm).

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLES

FIG. 6 illustrates average tensile strength of NDGA-treated collagenfibers that can be produced according to embodiments of the invention.In some embodiments, the fibers can be produced by the below described12 step process. The reference to the lower strength “NDGA fibers” inFIG. 6 refers to prior art fibers as described in U.S. Pat. No.6,565,960 and/or Koob and Hernandez, Material properties of polymerizedNDGA-collagen composite fibers: development of biologically based tendonconstructs, Biomaterials, 2002 January; 23 (1): 203-12.

-   1. Purify Type I collagen from 8-9 month old fetal bovine tendons as    previously described (Koob and Hernandez, Biomaterials 2002, supra).-   2. Dilute the purified collagen prep with 3% acetic acid to a final    concentration of 0.2% (weight/volume).-   3. Place the purified collagen prep in 3.2 mm diameter dialysis    tubes and dialyze 3 times against DI (di-ionized) water in a volume    ration of 60 to 1 for 40 minutes each time.-   4. Incubate in DI water for 36 hours at room temperature (25° C.)-   5. Place in fiber-forming buffer of 30 mM NaH₂PO₄, 140 mM NaCl in a    volume ratio of 60 to 1 for 16 hours at 37° C. This causes the    collagen to form a gel within the dialysis tubes.-   6. Extrude the collagen fiber gel into a water bath, hang from one    end and lift out of the water bath at a rate of 1 mm/min and allow    drying for at least 6 hours.-   7. Hydrate the dried fibers for 1 hr in at least 50 ml of buffer    (0.1 M NaH₂PO₄ pH 7.0) per gram of dry fiber.-   8. Dissolve 30 mg/ml NDGA in 0.4 N NaOH-   9. Add the dissolved NDGA solution to the buffer and fibers (use    3.33 mg NDGA per ml of 0.1 M NaH₂PO₄). Agitate for 16 hours-   10. Hang for 2 hrs with a 6.7 gram weight clamped to the bottom to    provide tension while drying.-   11. Place in 50 ml of 70% ethanol per gram of dry fiber and agitate    for 2 hrs of washing to remove any unreacted, soluble, NDGA    cross-linking intermediates.-   12. Dry again as per step 10 and repeat the NDGA treatment as per    steps 7 through 9 above.

FIG. 7 illustrates different donor or starting collagen materials,including bovine, caprine, porcine and echinoderm produced according tothe methods described herein and treated with NDGA having associatedaverage tensile strength using manufacturing methods described herein.In the case of the echinoderm collagen fibers, the collagen fibrils wereproduced by water extraction of the sea cucumber dermis producing intactnative fibrils according to published methods. See, Trotter, J. A.,Thurmond, F. A. and Koob, T. J. (1994) Molecular structure andfunctional morphology of echinoderm collagen fibrils. Cell Tiss. Res.275; 451-458.

NDGA treated collagen constructs have biocompatibility, suitablebiomechanical properties and the potential for biologic in-growth ofnative tissue for long-term stability.

The foregoing is illustrative of the present invention and is not to beconstrued as limiting thereof. Although a few exemplary embodiments ofthis invention have been described, those skilled in the art willreadily appreciate that many modifications are possible in the exemplaryembodiments without materially departing from the novel teachings andadvantages of this invention. Accordingly, all such modifications areintended to be included within the scope of this invention as defined inthe claims. The invention is defined by the following claims, withequivalents of the claims to be included therein.

That which is claimed:
 1. A method of manufacturing nordihydroguaiareticacid (NDGA) polymerized collagen fibers, comprising: (a) treatingcollagen with a solution comprising NDGA to provide NDGA-treatedcollagen; (b) drying the NDGA-treated collagen while holding theNDGA-treated collagen in tension for a period of time; (c) washing thedried NDGA-treated collagen in a solution to remove unreacted solubleNDGA cross-linking intermediates; (d) drying the NDGA-treated collagenwhile holding the NDGA-treated collagen in tension for a period of time;and repeating steps (a)-(d) at least once to produce high-strength NDGApolymerized collagen fibers, wherein the high-strength NDGA polymerizedcollagen fibers have an average tensile strength of at least about 150MPa.
 2. A method according to claim 1, wherein the washing step iscarried out using an ethanol solution.
 3. A method according to claim 1,wherein the washing step comprises agitating the NDGA-treated collagenin an ethanol solution for between about 1-4 hours.
 4. A methodaccording to claim 1, wherein the drying steps are carried out whilecollagen fibers associated with the NDGA-treated collagen are held intension for between about 1-5 hours.
 5. A method according to claim 4,wherein the collagen fibers are held in tension for about 2 hours duringthe drying steps.
 6. A method according to claim 1, wherein the dryingsteps include holding the NDGA-treated collagen fibers in tension with atensile force of between about 2 grams to about 10 grams.
 7. A methodaccording to claim 1, wherein the drying steps include clamping bottomportions of collagen fibers associated with the NDGA-treated collagen toa weight of between about 5-10 grams to apply a tensile force.
 8. Amethod according to claim 1, further comprising, after the steps arerepeated at least once, forming a bioprosthesis using the high-strengthNDGA polymerized fibers.
 9. A method according to claim 8, wherein thebioprosthesis is a ligament bioprosthesis that has a tensile strength ofbetween about 180-280 MPa, and a stiffness and dynamic flexibility thatmeets or exceeds that of a natural ligament.
 10. A method according toclaim 8, wherein the bioprosthesis has a tensile strength between about180-280 MPa, and a stiffness and dynamic flexibility that meets orexceeds that of a natural tendon.
 11. A method according to claim 10,wherein the NDGA polymerized collagen fibers have a dry diameter onaverage of about 0.08 mm and a wet diameter on average of about 0.13 mm.12. A method of organizing collagen before cross-linking, comprising:purifying donor collagen preparatory material; dialyzing the purifiedcollagen preparatory material at least three times against deionizedwater in a volume ratio of between about 30:1 to about 100:1, forbetween about 30-90 minutes to form a dialyzed collagen material; andforming a substantially clear gel using the dialyzed collagen materialthereby indicating improved organization of collagen fibrils.
 13. Amethod according to claim 12, wherein the dialyzing is carried out threetimes against deionized water in a volume ration of about 60 to 1 forabout 40 minutes.
 14. A method according to claim 12, further comprisingincubating the dialysized collagen material for at least about 24 hoursat a temperature of between about 15-30° C. to form incubated collagenmaterial; then placing the incubated collagen material in a fiberforming buffer to form a buffered collagen material; extruding thebuffered collagen material; cross-linking the buffered collagenmaterial; then forming polymerized collagen fibers.
 15. A methodaccording to claim 14, wherein the cross-linking is carried out usingNDGA, and wherein the forming step forms NDGA treated collagen fibershaving a dry diameter of about 0.08 mm, on average.